A conventional approach to tracking moving components in a sample is to determine changes relative to the background in order to identify regions which may be mobile components of interest. In fluorescence microscopy, the components of interest may be tagged by fluorescent labels or probes, making the task of distinguishing them from the background relatively straightforward.
Fluorescent molecules employed as labels typically have a specific excitation spectrum, being more strongly excited at some wavelengths and less strongly excited at others. They also have a specific emission spectrum, emitting more intensely at some wavelengths, and less intensely at others. The excitation and emission spectra may range from the ultraviolet to the infrared.
A wide range of fluorescent probes have been developed from chemical molecules, such as Rhodamine and Fluoroscein. Further fluorescent probes have been developed from molecules found in luminescent organisms, for example the Aequorea Jellyfish which has provided the Green Fluorescent Protein (GFP), and various corals providing DsRed and HcRed. These have been termed AFPs (Aequorea Fluorescent Proteins) and are described for example in J. Zhang, et al, Nature Reviews: Molecular Cell Biol., Vol 3, December 2002, pp 906-918; Y. A. Labas, Proc. Natl. Acad. Sci., 2002, Vol 99, pp 4256-4261; and M. V. Matz, Bioassay, Vol 24, pp 953-959.
The fluorescent probes may be associated with specific molecules of interest (DNA, RNA, proteins, carbohydrates, antibodies, etc). Alternatively they may be made to be sensitive to certain characteristics (ionic concentration, pH, voltage potential, temperature, the presence of a specific enzyme, the presence of specific enzyme substrates, force), altering their fluorescent properties according to these characteristics. These labels may be introduced into cells by passing through the cell membrane or by injection. Alternatively they may be formed internally as part of the normal functioning of the cell, in the case of the genetically encoded probes such as the AFPs.
In a known apparatus for imaging samples including fluorescent labels, a fluorescence microscope (FIG. 1) is fitted with an excitation light source which is capable of exciting one or more fluorescent probes at specific wavebands. The microscope is also fitted with suitable optical filters so that the light emitted from the probes may be observed at other wavebands. Examining the spatial and temporal distribution of light emitted provides information on the structure and dynamics of the sample.
The use of multiple labels provides information on the coincident localisation of labeled components, revealing, for example, the organization of the cytoskeleton of a cell. The fluorescence microscope is often fitted with an image acquisition system, comprising a light sensitive detector (sensitive from the ultraviolet to the infrared) such as a CCD camera or a combination of a scanner and a photomultiplier tube, and recording means such as a video recorder or computer system with a memory device, so that dynamic behaviour of the sample may be captured and analysed offline.
The system may employ a focus drive mechanism for altering the position of the imaging focus plane, thus allowing volumetric (XYZ) and volumetric time series (XYZT) data to be acquired. By selecting suitable excitation and/or emission wavebands and/or selecting suitable optical filter sets, volumetric multi-wavelength (XYWZ) and volumetric multi-wavelength time series (XYWZT) data may be acquired.
The microscope may also be fitted with additional apparatus to control the temperature, gas content and flow, introduce liquids, etc.
In order to gain a deeper understanding of the dynamic processes in a sample such as a living cell, an additional activating light beam may be provided (in many cases based on the excitation light source). The activating light beam may be directed to portions of the sample containing labels in such a manner that the intense light from this beam bleaches the label and reduces its fluorescence. By observing the subsequent development of fluorescence in this region, and elsewhere in the sample, information can be obtained on mechanisms of interaction and exchange of various labeled components (for example, as described in AxelRod, Biophys. J., 1977, Vol 18, pp 129-131; and Phair et al, Nature, 2000, Vol 404, pp 604-609).
Methods and apparatus for analysis of samples including fluorescent labels are disclosed in the present applicant's copending United Kingdom Patent Application No. 0419325.6, the contents of which are incorporated herein as reference material.
Moving components may often be distinguishable by their size, shape or trajectory, for example. However, this can be problematic if the components concerned are very similar in shape and size, or vary considerably in shape and size over time, or have unpredictable trajectories.
Existing approaches to addressing this problem include locating candidate particles and estimating the matches between frames. Examples are described in J. L. Barron, Fleet, D. J., and Beauchemin, S. (1994) Performance of optical flow techniques, International Journal of Computer Vision, 12(1):43-77; Nagel, H.-H. 1977, Analysing Sequences of TV-Frames: System Design Considerations, In Proc. Intern. Joint Conference on Artificial Intelligence, Cambridge, Mass., 626; Nagel, H.-H. 2000, Image Sequence Evaluation: 30 Years and Still Going Strong, In Proc. 15th Intern. Conf. Pattern Recognition, A. Sanfeliu, J. J. Villanueva, M. Vanrell, R. Alqu'ezar, J.-O. Eklundh, and Y. Aloimonos (Eds.), Vol. 1, 149-158. Los Alamitos, Calif.: IEEE Computer Society; M. Isard and A. Blake, Condensation—conditional density propagation for visual tracking, International Journal of Computer Vision 29(1), pp. 5-28, 1998; D. Comaniciu, V. Ramesh, and P. Meer, Kernel-Based Object Tracking, IEEE PAMI, vol. 25, no. 5, May 2003; Raffel M., Willert C., Kompenhans J (1998) Particle Image Velocimetry. Springer, Berlin; Stanislas, M., Kompenhans, J., Westerweel, J., Particle Image Velocimetry—Progress towards Industrial Application, Kluwer Academic Publishers, 2000.
Separate bleach and imaging scanning techniques are described in UK Patent Specification No. 2369739, and “Beam Control in a Scanning Microscope”, J. Engelhardt/Leica, 5 Jun. 2002.